The Cre-loxP system of bacteriophage P1 has been shown to efficiently generate recombinant nucleic acids which may encode hybrid proteins. This system requires only two well-characterized components: a 38 kDa recombinase protein, Cre, and a 34 bp loxP target sequence (Kilby, N. J., M. R. Snaith, and J. A. H. Murray. 1993, Trends Genet. 9: 413-421.). Cre binds to the two 13 bp inverted repeats of loxP and catalyzes precise recombination between the asymmetric 8 bp core regions of two loxP sites (Kilby, N. J., M. R. Snaith, and J. A. H. Murray. 1993, Trends Genet. 9: 413-421.). Recombination between two parallel sites, as defined by the core region, results in excision of intervening sequences producing two recombination products each containing one loxP site, whereas recombination between anti-parallel sites inverts the bracketed fragment. Intermolecular recombination between loxP sites on separate plasmids results in integration of sequences bracketed by loxP sites. The Cre-loxP system has been shown to function in both bacteria (Sternberg, N., B. Sauer, R. Hoess, and K. Abremski. 1986, J. Mol. Biol. 187: 197-212.) and eukaryotic cells (Sauer, B., and N. Henderson. 1989, Nucl. Acids Res. 17: 147-161.) and has been exploited for the excision (Sauer, B., and N. Henderson. 1989, Nucl. Acids Res. 17: 147-161.) and the integration of fragments in cellular and viral genomes (Sauer, B., and N. Henderson. 1990, The New Biologist 2: 441-449.; Sauer, B., M. Whealy, A. Robbins, and L. Enquist. 1987, Proc. Natl. Acad. Sci. USA 84: 9108-9112.). The use of Cre in cell-free systems for construction of recombinant vectors has also been reported (Gage, P. J., B. Sauer, M. Levine, and J. C. Glorioso. 1992, J. Virol. 66: 5509-5515; Sauer, B., M. Whealy, A. Robbins, and L. Enquist. 1987, Proc. Natl. Acad. Sci. USA 84: 9108-9112.).
Cre-loxP based recombination has further been used successfully for tissue-specific gene expression or deletion in transgenic mice (Pichel, J. G., M. Lakso, and H. Westphal. 1993, Oncogene 8: 3333-3342.; Gu, H., J. D. Marth, P. C. Orban, H. Mossmann, and K. Rajewsky. 1994, Science 265: 103-106.). In the latter cases, the recombinase was delivered by transfection (Gu, H., J. D. Marth, P. C. Orban, H. Mossmann, and K. Rajewsky. 1994, Science 265: 103-106.) or micro injection (Araki, K., M. Araki, J.-I. Miyazaki, and P. Vassalli. 1995, Proc. Natl. Acad. Sci. USA 92: 160-164.; Pichel, J. G., M. Lakso, and H. Westphal. 1993, Oncogene 8: 3333-3342.) of Cre encoding plasmids into ES cells or fertilized eggs.